β-Barrel domain swapping in α-hemolysin enables enhanced single-molecule biomolecule sensing
Biological nanopores are powerful platforms for single-molecule analysis, yet rational strategies to tune their transport and sensing properties remain limited. Here we present a modular engineering approach based on {beta}-barrel domain swapping to reprogram the function of the prototypical nanopore -hemolysin. By replacing its native {beta}-barrel with {beta}-barrel domains from diverse pore-forming toxins, we generate a series of chimera nanopores that retain oligomerization capability while exhibiting reduced membrane-permeabilizing activity. Electrophysiological measurements show that selected chimera pores form stable, conductive channels with distinct ion transport properties. Notably, the HL_NetB chimera displays stable conductance, enhanced electroosmotic flow, and improved performance in nucleic acid and protein sensing. Single-molecule experiments demonstrate that this chimera markedly slows the translocation of single-stranded DNA, enabling discrimination by length and sequence, improves the resolution of intrinsically disordered proteins such as -synuclein, and enhances sensitivity to RNA conformational changes. Together, these results establish {beta}-barrel domain swapping as a general and effective strategy for engineering biological nanopores with tailored single-molecule sensing capabilities.